Device and method for photoactivation

ABSTRACT

Methods and compositions are described for treating contaminants in material intended for in vivo use, and in particular blood and blood products for human use. Contaminants in blood cell preparations are inactivated prior to long term storage and transfusion. Inactivation is accomplished using a device having a unique temperature control design.

[0001] The present application is a continuation of Ser. No. 10/180,428filed Jun. 15, 2002, which is a continuation of Ser. No. 09/349,646filed Jul. 8, 1999, which is a continuation of Ser. No. 8/664,992 filedJun. 13, 1996, which is a continuation of Ser. No. 08/380,154 filed Jan.30, 1995, which is a continuation of Ser. No. 08/150,940 filed Nov. 10,1993, which is a continuation-in-part of application Ser. No.07/844,790, filed Mar. 2, 1992 which issued as U.S. Pat. No. 5,288,605.

FIELD OF THE INVENTION

[0002] The invention generally relates to a device and method forphotoactivating new and known compounds.

BACKGROUND

[0003] Whole blood collected from volunteer donors for transfusionrecipients is typically separated into its components: red blood cells,platelets, and plasma. Each of these fractions is individually storedand used to treat a multiplicity of specific conditions and diseasestates. For example, the red blood cell component is used to treatanemia; the concentrated platelet component is used to control bleeding;and the plasma component is used frequently as a source of ClottingFactor VIII for the treatment of hemophilia.

[0004] Ideally, all blood cell preparations should be from freshly drawnblood and then immediately transfused to the recipient. However, thelogistics of operating a blood donor center preclude this possibility inthe vast majority of cases. Transfusions are needed day and night and itis difficult, if not impossible, to arrange for donor recruiting atunusual hours. Consequently, modern blood donor centers must use storedblood products.

[0005] In the United States, blood storage procedures are subject toregulation by the government. The maximum storage periods for the bloodcomponents collected in these systems are specifically prescribed. Forexample, whole blood components collected in an “open” (i.e.non-sterile) system must, under governmental rules, be transfused withintwenty-four hours and in most cases within six to eight hours. Bycontrast, when whole blood components are collected in a “closed” (i.e.sterile) system the red blood cells can be stored up to forty-two days(depending upon the type of anticoagulant and storage medium used) andplasma may be frozen and stored for even longer periods.

[0006] Murphy and Gardner, New Eng.J.Med. 280:1094 (1969), demonstratedthat platelets stored as platelet-rich plasma (PRP) at 22° C. possesseda better in vivo half-life than those stored at 4° C. Thus, moreacceptable platelet concentrates could be transfused after storage atroom temperature. Until recently, the rules allowed for plateletconcentrate storage at room temperature for up to seven days (dependingupon the type of storage container). However, it was recognized that theincidence of bacterial growth and subsequent transfusion reactions inthe recipient increased to unacceptable levels with a seven day oldplatelet concentrate. Platelet concentrates may now be stored for nomore than five days.

[0007] Blood bags used for platelet concentrate preparation are inthemselves sterile, as are the connected satellite bags. One mightbelieve, therefore, that it is a relatively simple matter to keep theblood preparation sterile during the manipulations needed to concentratethe platelets. However, bacteria can be introduced by at least twodifferent means. First, if the donor is experiencing a mild bacteremia,the blood will be contaminated, regardless of the collection or storagemethod. Adequate donor histories and physicals will decrease but noteliminate this problem. See B. J. Grossman et al., Transfusion 31:500(1991). A second, more pervasive source of contamination is thevenepuncture. Even when “sterile” methods of skin preparation areemployed, it is extremely difficult to sterilize the crypts around thesweat glands and hair follicles. During venepuncture, this contaminatedskin is often cut out in a small “core” by a sharp needle. This core canserve to “seed” the blood bag with bacteria that may grow and become arisk to the recipient.

[0008] Indeed, many patients requiring platelet transfusions lackhost-defense mechanisms for normal clearing and destruction of bacteriabecause of either chemotherapy or basic hematological disease. Thegrowth of even seemingly innocuous organisms in stored platelets can,upon transfusion, result in recipient reaction and death. See e.g. B. A.Myhre JAMA 244:1333 (1980). J. M. Heal et al. Transfusion 27:2 (1987).

[0009] The reports assessing the extent of contamination in plateletshave differed in their methods, sample size, and bacterial detectionschemes. D. H. Buchholz, et al., Transfusion 13:268 (1973) reported anoverall level of platelet contamination of 2.4% when a large (>1000bags) sample was examined and extensive measures were taken forbacterial culturing. While some units were heavily contaminated afterjust 24 hours of storage, the incidence as a whole varied according tothe age of the concentrate and increased with the widespread practice ofpooling individual units; over 30% of pools were contaminated at 3 days.See also D. H. Buccholz, et al., New Eng. J. Med. 285:429 (1971). Whileother clinicians suggest lower numbers, recent studies indicate thatseptic platelet transfusions are significantly underreported. See e.g.J. F. Morrow et al. JAMA 266:555 (1991).

[0010] Pre-culturing platelets is not a solution to the bacterialcontamination problem. The culture assay takes 48 hours to detectgrowth. Holding platelet units for an additional two days to await theresults of the assay would create, ironically, a smaller margin ofsafety. See Table 2 in J. F. Morrow et al. JAMA 266:555 (1991). Whileheavily contaminated units would be detected at the outset, lightlycontaminated units would be allowed to grow for two days. Older andpotentially more contaminated units would end up being transfused.

[0011] Washing the blood cells (e.g. with saline) or filtering thebacteria are also not practical solutions. These techniques are timeconsuming and inefficient, as they can reduce the number of viable bloodcells available for transfusion. Most importantly, they typicallyinvolve an “entry” into the storage system. Once an entry is made in apreviously closed system, the system is considered “opened,” andtransfusion must occur quickly, regardless of the manner in which theblood was collected and processed in the first place.

[0012] Nor are antibiotics a reasonable solution. Contamination occursfrom a wide spectrum of organisms. Antibiotics would be needed to coverthis spectrum. Many recipients are allergic to antibiotics. In addition,there is an every increasing array of drug-resistant strains of bacteriathat would not be inactivated.

[0013] There has been interest recently in inactivation of pathogens inblood using photoreactive compounds, such as psoralens. Psoralens aretricyclic compounds formed by the linear fusion of a furan ring with acoumarin. Psoralens can intercalate between the base pairs ofdouble-stranded nucleic acids, forming covalent adducts to pyrimidinebases upon absorption of long wave ultraviolet light (UVA). G. D. Ciminoet al., Ann. Rev. Biochem. 54:1151 (1985). Hearst et al., Quart. Rev.Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to apsoralen-pyrimidine monoadduct and on the opposite strand, absorption ofa second photon can lead to formation of a diadduct which functions asan interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058(1977). S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294(1982). J. Tessman et al., Biochem. 24:1669 (1985). Hearst et al., U.S.Pat. Nos. 4,124,589, 4,169,204, and 4,196,281, hereby incorporated byreference.

[0014] Psoralens have been shown to inactivate viruses in some bloodproducts. See H. J. Alter et al., The Lancet (ii:1446) (1988). L. Lin etal., Blood 74:517 (1989). G. P. Wiesehahn et al., U.S. Pat. Nos.4,727,027 and 4,748,120, hereby incorporated by reference, describe theuse of a combination of 8-methoxypsoralen (8-MOP) and irradiation. Theyshow that 300 ug/ml of 8-MOP together with one hour or more ofirradiation with ultraviolet light can effectively inactivate viruses.However, these treatment conditions cause harm to the blood productbecause of energy transfer. Their approach is only feasible if thedamage to cells is specifically suppressed by limiting the concentrationof molecular oxygen, a difficult and expensive process.

[0015] Isopsoralens, like psoralens, are tricyclic compounds formed bythe fusion of a furan ring with a coumarin. See Baccichetti et al., U.S.Pat. No. 4,312,883. F. Bordin et al., Experientia 35:1567 (1979). F.Dall'Acqua et al., Medeline Biologie Envir. 9:303 (1981). S. Caffieri etal., Medecine Biologie Envir. 11:386 (1983). F. Dall'Acqua et al.,Photochem Photobio. 37:373 (1983). G. Guiotto et al., Eur. J. Med.Chem-Chim. Ther. 16:489 (1981). F. Dall'Acqua et al., J. Med. Chem.24:178 (1984). Unlike psoralens, the rings of isopsoralen are notlinearly annulated. While able to intercalate between the base pairs ofdouble-stranded nucleic acids and form covalent adducts to nucleic acidbases upon absorption of longwave ultraviolet light, isopsoralens, dueto their angular geometry, normally cannot form crosslinks with DNA. Seegenerally, G. D. Cimino et al., Ann. Rev. Biochem. 54:1151 (1985).

[0016] There are devices presently employed which emit ultravioletradiation for activating psoralens and other photoactivated compounds.U.S. Pat. No. 5,184,020, to Hearst, et al., discloses such a device forphotoactivating psoralens. However, the disclosed device is structuredfor the irradiation of samples in tube like vessels. It does notdisclose a device for use on blood bags. Further, although the patentdiscloses a cooling system for the irradiated samples, this system wouldnot work for blood bags because it depends on the circulation of fluidaround the sample vessels.

[0017] Other devices are not appropriate for activating psoralens, butcan be used for other purposes with blood bags. For example, U.S. Pat.Nos. 4,726,949 and 4,866,282, to Miripol, disclose such an irradiationdevice for use in preventing alloimmunization. This device is notpractical for use in laboratories which will process large quantities ofblood for sterilization. The device only supports one blood container,which would bottleneck the processing of blood. (See FIG. 1, ref. no.10, of either Miripol patent). Further, it provides radiation ofwavelength from 280 to 320 nanometers, including the 313 band, (seeclaim 1 of the '282 patent) at which nucleic acids absorb radiation andcould be damaged. The UVB range can also destroy platelet function. TheMiripol patents state that UV-A range sources “do not provide goodreduction of the lymphocyte alloimmunization effect.” Column 2, line61-64, of '949. Finally, the Miripol patents disclose the use of onlyone means for cooling the system during irradiation, an exhaust fan. Thegoal in those patents is to maintain the heat at 31 degrees C. or less.Column 3, line 44-46. However, platelets are currently stored at 22-24degrees C. G. Stack and L. Snyder, “Storage of Platelet Concentrate,”Blood Separation and Platelet Fractionation, pp. 9-125 (1991 Wiley-Liss,Inc.)

[0018] Last, there are devices disclosed which would neither beappropriate for activating psoralens nor for other uses on bloodproducts. U.S. Pat. No. 4,421,987, to Herold, discloses an apparatus forirradiating dental objects which employs radiation in the spectral rangeof 400 to 500 nm, for bleaching treatment of dental parts. The device isfitted with a selective reflector which reflects from the totalradiation emitted by the lamp only the spectral portion lying in thedesired spectral range (approximately 400 to 500 nm) while transmittingor passing the portion of the radiation lying outside this desiredspectral range. The device also has a temperature control system,employing the combination of a blower with an absorption filter which,like the reflector, removes radiation outside of the desired spectralrange. This apparatus is not suited for the present purpose of aphotodecontamination treatment, because it is designed for use withwavelengths of light which are damaging to some blood components, whileit removes wavelengths necessary to activate certain photoreactivecompounds. Further, it is not equipped with a temperature maintainingsystem which would keep the temperature of blood samples low enough toprevent damage.

[0019] In sum, there is a need for a means of inactivating bacteria inblood components prior to storage and transfusion in a way that lendsitself to use in a closed system, such as a system of blood bags. Thisapproach must be able to handle a high volume of blood and a variety oforganisms while efficiently controlling the temperature and avoidingharm to the blood product or the transfusion recipient.

SUMMARY OF THE INVENTION

[0020] The present invention relates to a device and method forphotoactivating new and known compounds. The present invention furthercontemplates devices for binding new and known compounds to nucleicacid. Specifically, the present invention contemplates a device forphotoactivating new and known compounds so that they bind to andinactivate bloodborne pathogens. In accordance with the presentinvention, a nucleic acid binding compound is selectively employed totreat contamination by microorganisms.

[0021] In one embodiment, the present invention contemplates: aphotoactivation device for inactivating pathogens in blood products,comprising: a housing; means for providing electromagnetic radiation tocause activation of at least one photoreactive compound, containedwithin said housing; means for supporting a plurality of blood bags,containing said photoreactive compound, at a fixed distance from saidradiation providing means during said activation, comprising a lowerultraviolet light transparent plate assembly within said housing, uponwhich said blood bags can rest; and an upper ultraviolet lighttransparent plate assembly, positioned above said lower plate assembly,said upper and lower plate assemblies defining a channel, closed offfrom significant exchange with air originating from outside said housingduring irradiation, in which air can be circulated to cool said bloodbags. In another embodiment, said lower plate assembly comprises a topand a bottom plate and an air circulation chamber between said top andbottom plates, open to said channel to allow air exchange between saidair circulation chamber and said channel.

[0022] In a preferred embodiment, the device further comprises: firsttemperature maintaining means, comprising: means for blowing air fromoutside, through said housing, between said irradiation providing meansand said plate assemblies, positioned within and adjacent to saidhousing, for cooling said irradiation providing means; and secondtemperature maintaining means, positioned within said housing, forcirculating cooled air through said air circulation chamber and saidchannel, comprising: a heat exchanger, between said plate assemblies,for absorbing heat from air present in said housing, and; means forcirculating air, positioned in a fixed relationship to said heatexchanger. In one embodiment, said heat exchanger comprises a conduithaving an inlet port and an outlet port so that temperature controlliquid may enter and exit. In a preferred embodiment, said upper andlower plate assemblies are separated by between approximately 1 and 10cm. However, it is preferred that when said blood bags rest upon saidlower plate assembly, said upper plate assembly does not contact saidblood bags.

[0023] Because of the benefits of rapid processing, in one contemplatedembodiment said lower plate assembly is of dimensions sufficient tosupport six of said blood bags. Said blood bag supporting means mayfurther comprise means to position a plurality of attachments connectedto said blood bags, so that said attachments do not significantly reducethe intensity of radiation to said blood bags, including tubing fortransferring a blood product into or out of said blood bags and a bloodproduct storage bag. The device may further comprise means for shakingsaid blood bag supporting means, positioned adjacent to said blood bagsupporting means, for providing mixing of a sample in a blood bag duringirradiation. The present invention contemplates that said lower plateassembly has a ridged upper surface to maintain the position of saidblood bags during shaking.

[0024] In a preferred embodiment, said housing comprises material whichblocks said electromagnetic radiation so that users are shielded fromsaid electromagnetic radiation during said activation. Also contemplatedis a means for controlling said radiation providing means, which maycomprise a plurality of detectors, positioned around said radiationproviding means, to measure said electromagnetic radiation; and afeedback control, connected to said detectors, which shuts off saidradiation providing means at a desired output of radiation detected bysaid detectors. Preferably, the intensity of radiation provided by saidradiation providing means is at least 15 mW/cm², and said radiationproviding means has a high end wavelength cutoff above 400 nanometers.Additionally, it is contemplated that the upper and lower plateassemblies are comprised of material which filters said electromagneticradiation to provide a low end wavelength cutoff below 320 nanometers.

[0025] The radiation providing means may further comprise a top bank anda bottom bank of light sources, said top bank being located above saidupper plate assembly, and said bottom bank being located below saidlower plate assembly. Reflecting means, adjacent to said top bank andsaid bottom bank of light sources, are also contemplated, which reflectelectromagnetic radiation from said light sources toward said blood bagsupporting means.

[0026] In an alternative embodiment of the present invention, aphotoactivation device is contemplated for treating photoreactivecompounds, comprising: an opaque housing; means for providingelectromagnetic radiation to cause activation of at least onephotoreactive compound, within said housing; means for supporting aplurality of blood bags at a fixed distance from said radiationproviding means during said activation, comprising a lower ultravioletlight transparent plate assembly, within said housing, upon which saidblood bags can rest; an upper ultraviolet light transparent plateassembly, positioned above said lower plate assembly, said upper andlower plate assembly defining a channel, closed off from significantexchange with air originating from outside said housing duringirradiation, through which air can be circulated to cool said bloodbags; first temperature maintaining means, comprising: means for blowingair from outside, through said housing, between said irradiationproviding means and said plate assemblies, positioned within andadjacent to said housing, for cooling said irradiation providing means;and second temperature maintaining means, positioned within saidhousing, for circulating cooled air through said channel, comprising: aheat exchanger, between said plate assemblies, for absorbing heat fromair present in said housing; and means for circulating air, positionedin a fixed relationship to said heat exchanger, for circulating cooledair from said heat exchanger through said channel. In one embodiment,said heat exchanger comprises a conduit having an inlet port and anoutlet port so that temperature control liquid may enter and exit. Saidlower plate assembly may be comprised of a top and a bottom plate, andan air circulation chamber between said top and bottom plates. In thisembodiment, said means for circulating air circulates air through saidair circulation chamber.

[0027] It is contemplated that said upper and lower plate assemblies areseparated by between approximately 1 and 10 cm. However, in a preferredembodiment, when said blood bags rest upon said lower plate assembly,said upper plate assembly does not contact said blood bags. Alsocontemplated is a lower plate assembly of dimensions sufficient tosupport six of said blood bags. It is contemplated that the blood bagsupporting means further comprises means to position a plurality ofattachments connected to said blood bags, so that said attachments donot significantly reduce the intensity of radiation to said blood bags.Some such attachments comprise tubing for transferring a blood productinto or out of said blood bags and a blood product storage bag. Thepresent invention further contemplates means for shaking said blood bagsupporting means, positioned adjacent to said blood bag supportingmeans, for providing mixing of a sample in a blood bag duringirradiation. In one embodiment, said lower plate assembly has a ridgedupper surface to maintain the position of said blood bags during saidshaking.

[0028] The present invention contemplates a photoactivation devicecomprising means for controlling said radiation providing means. Themeans for controlling said radiation providing means may comprise: aplurality of detectors, positioned around said radiation providingmeans, to measure said electromagnetic radiation; and a feedbackcontrol, connected to said detectors, which shuts off said radiationproviding means at a desired output of radiation detected by saiddetectors. In a preferred embodiment, the intensity of radiationprovided by said radiation providing means is at least 15 mW/cm² andsaid radiation providing means has a high end wavelength cutoff above400 nanometers.

[0029] In one embodiment, said plate assemblies are comprised ofmaterial which removes blood product damaging wavelengths of radiationfrom said electromagnetic radiation. Specifically, it is contemplatedthat said material filters said electromagnetic radiation to provide alow end wavelength cutoff below 320 nanometers. It is also contemplatedthat said radiation providing means comprises a top bank and a bottombank of light sources, said top bank being located above said upperplate assembly, and said bottom bank being located below said lowerplate assembly. Reflecting means may be positioned adjacent to said topbank and said bottom bank of light sources, which reflectelectromagnetic radiation from said light sources toward said blood bagsupporting means.

[0030] The present invention also contemplates a method forphotoactivating photoreactive compounds, comprising: supporting aplurality of blood bags, containing one or more photoreactive compounds,at a fixed distance from a fluorescent source of electromagneticradiation; irradiating said plurality of blood bags simultaneously withelectromagnetic radiation having a wavelength cutoff at approximately320 nm, from said fluorescent source to cause activation of at least oneof said photoreactive compounds; and maintaining the temperature of saidblood bags at approximately room temperature during said activation, bycooling air and circulating cooled air around said blood bags in aclosed system. Preferably, the fluorescent source of electromagneticradiation delivers an intensity of electromagnetic radiation greaterthan 1 mW/cm² to said blood bags.

DESCRIPTION OF THE FIGURES

[0031]FIG. 1 is a perspective view of one embodiment of the device ofthe present invention in the closed position.

[0032]FIG. 2 is a cross-sectional view of the device shown in FIG. 1, inthe open position, along the lines of 2-2.

[0033]FIG. 3 is a cross-sectional view of the device shown in FIG. 1along the lines of 3-3.

[0034]FIG. 4 is a cross-sectional view of the device shown in FIG. 1along the lines of 4-4.

[0035]FIG. 5 schematically shows the decontamination approach of thepresent invention applied specifically to blood products.

[0036]FIG. 6 is a graph showing the photoaddition of 8-methoxypsoralento nucleic acid.

[0037]FIG. 7 is a graph showing the degradation of 8-methoxypsoralen(8-MOP) compared to that of 4′-aminomethyl-4,5′,8-trimethylpsoralen(AMT), as measured by HPLC.

DESCRIPTION OF THE INVENTION

[0038] The present invention relates to a device and method forphotoactivating new and known compounds.

[0039] As noted previously, whole blood is collected and typicallyseparated into red blood cells, platelets, and plasma. Each of thesefractions are individually stored under specific conditions prior to invivo use. In many cases, the extent of contamination is related to thestorage time because of growth. A process that inactivatedmicroorganisms at the time of blood collection would be expected toprevent growth during storage.

Table 1. Photoreactive Compounds

[0040] Actinomycins

[0041] Anthracyclinones

[0042] Anthramycin

[0043] Benzodipyrones

[0044] Fluorenes and fluorenones

[0045] Furocoumarins

[0046] Mitomycin

[0047] Monostral Fast Blue

[0048] Norphillin A

[0049] Many organic dyes not specifically listed

[0050] Phenanthridines

[0051] Phenazathionium Salts

[0052] Phenazines

[0053] Phenothiazines

[0054] Phenylazides

[0055] Quinolines

[0056] Thiaxanthenones

[0057] “Photoactivation compounds” (or “photoreactive compounds”)defines a family of compounds that undergo chemical change in responseto electromagnetic radiation (Table 1). One species of photoreactivecompounds described herein is commonly referred to as the furocoumarins.The furocoumarins belong to two main categories: 1) psoralens[7H-furo(3,2-g)-(1)-benzopyran-7-one, or .-lactone of6-hydroxy-5-benzofuranacrylic acid], which are linear:

[0058] and in which the two oxygen residues appended to the centralaromatic moiety have a 1, 3 orientation, and further in which the furanring moiety is linked to the 6 position of the two ring coumarin system,and 2) the isopsoralens [2H-furo(2,3-h)-(1)-benzopyran-2-one, or.-lactone of 4-hydroxy-5-benzofuranacrylic acid], which are angular:

[0059] in which the two oxygen residues appended to the central aromaticmoiety have a 1, 3 orientation, and further in which the furan ringmoiety is linked to the 8 position of the two ring coumarin system.Psoralen derivatives are derived from substitution of the linearfurocoumarin at the 3, 4, 5, 8, 4′, or 5′ positions, while isopsoralenderivatives are derived from substitution of the angular furocoumarin atthe 3, 4, 5, 6, 4′, or 5 positions.

[0060] In one embodiment, the present invention contemplatesinactivating blood products after separation but before storage. In thisembodiment, a nucleic acid binding compound is selectively employed totreat contamination by microorganisms.

[0061] In one embodiment, the nucleic acid binding compound is selectedfrom the group comprising furocoumarins. In a preferred embodiment, thefurocoumarin is a psoralen or isopsoralen.

[0062] The inactivation method of the present invention provides amethod of inactivating single cell and multicellular organisms, and inparticular, bacteria, fungi, mycoplasma and protozoa. In contrast toprevious approaches, the method of the present invention does not causeharm to the blood product. There is no significant damage to cells and,therefore, no need to limit the concentration of molecular oxygen.

[0063] The present invention contemplates using much lowerconcentrations of nucleic acid binding compounds than previouslyemployed. For example, the present invention contemplates using 8-MOP atconcentrations of 30 ug/ml or less. Indeed, a preferred concentration of8-MOP for bacterial decontamination in platelet concentrates is 3 ug/mlor less, i.e. a one hundred-fold lower concentration than employed by G.P. Wiesehahn et al., supra.

[0064] The present invention, furthermore, contemplates using much lowerdoses of irradiation than previously described. This is accomplishedwith lower intensity irradiation sources, with wavelength cutoff filters(see below), and/or shorter irradiation times. In a preferredembodiment, the time of irradiation is variable and controlled from 1second to 99 minutes, in one second increments.

[0065] While it is not intended that the present invention be limited bythe theory of inactivation, the use of lower compound concentrations andirradiation doses comes from an understanding that, where the presentinvention is applied to the decontamination of a single cell ormulticellular organism (as opposed to a virus), a lower level of nucleicacid binding will achieve inactivation. In addition, it is recognizedthat it is not essential that inactivation be complete. That is to say,partial inactivation will be adequate as long as the viable portion isunable, within the storage period, to grow to levels sufficient to causedisease.

[0066] To appreciate that, in any given case, an inactivation method mayor may not achieve complete inactivation, it is useful to consider aspecific example. A bacterial culture is said to be sterilized if analiquot of the culture, when transferred to a fresh culture plate andpermitted to grow, is undetectable after a certain time period. The timeperiod and the growth conditions (e.g. temperature) define an“amplification factor”. This amplification factor along with thelimitations of the detection method (e.g. visual inspection of theculture plate for the appearance of a bacterial colony) define thesensitivity of the inactivation method. A minimal number of viablebacteria must be applied to the plate for a signal to be detectable.With the optimum detection method, this minimal number is 1 bacterialcell. With a suboptimal detection method, the minimal number ofbacterial cells applied so that a signal is observed may be much greaterthan 1. The detection method determines a “threshold” below which themethod appears to be completely effective (and above which the methodis, in fact, only partially effective).

[0067] This interplay between the amplification factor of an assay andthe threshold that the detection method defines, can be illustrated. Forexample, bacterial cells can be applied to a plate; the detection methodis arbitrarily chosen to be visual inspection. Assume the growthconditions and time are such that an overall amplification of 10⁴ hasoccurred. The detectable signal will be proportional to the number ofbacterial cells actually present after amplification. For calculationpurposes, the detection threshold is taken to be 10⁶ cells; if fewerthan 10⁶ cells are present after amplification, no cell colonies arevisually detectable and the inactivation method will appear effective.Given the amplification factor of 10⁴ and a detection threshold of 10⁶,the sensitivity limit would be 100 bacterial cells; if less than 100viable bacterial cells were present in the original aliquot of thebacterial culture after the sterilization method is performed, theculture would still appear to be sterilized.

[0068] Such a situation is common for bacterial growth assays. Thesensitivity of the assay is such that viable bacterial cells are presentbut the assay is unable to detect them. This may explain, at least inpart, the variability in results obtained by researchers attempted todetermine the extent of bacterial contamination of blood products. SeeD. H. Buchholz, et al., Transfusion 13:268 (1973), wherein suchvariability is discussed.

[0069] It should be noted that, in many countries, contamination ofblood products by cellular organisms is more pervasive and, therefore,more serious than viral contamination. For example, in South America,the most important blood-borne organism is T. cruzi, which is theetiologic agent of Chagas disease. Approximately 16-18 million peopleare infected in the Americas (including 11% of the population of Chile).It is contemplated that the decontamination method of the presentinvention is well-suited for inactivation of this protozoa.

[0070] The present invention contemplates devices and methods forphotoactivation and specifically, for activation of photoreactivenucleic acid binding compounds. The present invention contemplatesdevices having an inexpensive source of electromagnetic radiation thatis integrated into a unit. In general, the present inventioncontemplates a photoactivation device for treating photoreactivecompounds, comprising: a) means for providing appropriate wavelengths ofelectromagnetic radiation to cause activation of at least onephotoreactive compound; b) means for supporting a plurality of bloodproducts at a fixed distance from the radiation providing means duringactivation; and c) means for maintaining the temperature of the bloodproducts within a desired temperature range during activation. Thepresent invention also contemplates methods, comprising: a) supporting aplurality of blood product containers, containing one or morephotoreactive compounds, at a fixed distance from a fluorescent sourceof electromagnetic radiation; b) irradiating the plurality of bloodproducts simultaneously with said electromagnetic radiation to causeactivation of at least one photoreactive compound; and c) maintainingthe temperature of the blood products within a desired temperature rangeduring activation.

[0071] The present invention contemplates devices and methods forphotoactivation and specifically, for inactivation of pathogenscontaminating blood products by activation of photoreactive compounds.The major features of one embodiment of the device of the presentinvention involve: A) an inexpensive source of ultraviolet radiation ata fixed distance from the means for supporting the sample vessels, B)rapid photoactivation, C) large sample processing, D) temperaturecontrol of the irradiated samples, E) inherent safety and F) samplecontainers.

A. Electromagnetic Radiation Source

[0072] A preferred photoactivation device of the present invention hasan inexpensive source of ultraviolet radiation at a fixed distance fromthe means for supporting the sample vessels. Ultraviolet radiation is aform of energy that occupies a portion of the electromagnetic radiationspectrum (the electromagnetic radiation spectrum ranges from cosmic raysto radio waves). Ultraviolet radiation can come from many natural andartificial sources. Depending on the source of ultraviolet radiation, itmay be accompanied by other (non-ultraviolet) types of electromagneticradiation (e.g. visible light).

[0073] Particular types of ultraviolet radiation are herein described interms of wavelength. Wavelength is herein described in terms ofnanometers (“nm”; 10⁻⁹ meters). For purposes herein, ultravioletradiation extends from approximately 180 nm to 400 nm. When a radiationsource, by virtue of filters or other means, does not allow passage ofradiation with wavelengths shorter than a particular wavelength (e.g.320 nm), it is said to have a low end “cutoff” at that wavelength (e.g.“a short wavelength cutoff at 320 nanometers”). Similarly, when aradiation source allows only passage of radiation with wavelengthsshorter than a particular wavelength (e.g. 360 nm), it is said to have ahigh end “cutoff” at that wavelength (e.g. “a long wavelength cutoff at360 nanometers”).

[0074] For any photochemical reaction it is desired to eliminate or atleast minimize any deleterious side reactions. Some of these sidereactions can be caused by the excitation of endogenous chromophoresthat may be present during the photochemical activation procedure. In asystem where only nucleic acid and psoralen are present, the endogenouschromophores are the nucleic acid bases themselves. Restricting theactivation process to wavelengths greater than 320 nm minimizes directnucleic acid damage since there is very little absorption by nucleicacids at wavelengths longer than 313 nm.

[0075] In blood products, the nucleic acid is typically present togetherwith additional biological chromophores. If the biological fluid is justprotein, the 320 nm short wavelength cutoff will be adequate forminimizing side reactions (aromatic amino acids do not absorb at shorterwavelengths than 320 nm). If the biological fluid includes cells and/orcellular constituents, there will be many other chromophores, includinghemes and flavins.

[0076] Hemes are abundant in blood products where they arise from thelysis of red cells. Flavins, like hemes, are required for metabolicrespiration. Both of these endogenous chromophores will cause damage tocells if excited by photoirradiation.

[0077] Hemes have three principle absorption bands: two are in the redregion of the visible spectrum; the other is centered about 400 nm.Flavins have two principle absorption peaks: one at 450 nm and the otherat 370 nm.

[0078] In view of the presence of these endogenous chromophores in bloodproducts, it is intended that in one embodiment of the device of thepresent invention the device is designed to allow for irradiation withina small range of specific and desirable wavelengths, and thus avoiddamage to cells caused by energy transfer. The preferred range ofdesirable wavelengths is between 320 and 350 nm.

[0079] Some selectivity can be achieved by choice of commercialirradiation sources. For example, while typical fluorescent tubes emitwavelengths ranging from 300 nm to above 400 nm (with a broad peakcentered around 360 nm), BLB type fluorescent lamps are designed toremove wavelengths longer than 400 nm. This, however, only provides along wavelength cutoff.

[0080] In a preferred embodiment, the device of the present inventioncomprises an additional filtering means. In one embodiment, thefiltering means comprises a glass cut-off filter, such as a piece ofCobalt glass. In another embodiment, the filtering means comprises aliquid filter solution that transmit only a specific region of theelectromagnetic spectrum, such as an aqueous solution of Co(No₃)₂. Thissalt solution yields a transmission window of 320-400 nm. In a preferredembodiment, the aqueous solution of Co(No₃)₂ is used in combination withNiSO₄ to remove the 365 nm component of the emission spectrum of thefluorescent or arc source employed. The Co—Ni solution preserves itsinitial transmission remarkably well even after tens of hours ofexposure to the direct light of high energy sources.

[0081] It is not intended that the present invention be limited by theparticular filter employed. Several inorganic salts and glasses satisfythe necessary requirements. For example, cupric sulfate is a most usefulgeneral filter for removing the infra-red, when only the ultraviolet isto be isolated. It offers stability in intense sources. Other salts areknown to one skilled in the art. Aperture or reflector lamps may also beused to achieve specific wavelengths and intensities.

[0082] When ultraviolet radiation is herein described in terms ofirradiance, it is expressed in terms of intensity flux (milliwatts persquare centimeter or “mW/cm^(2”)). “Output” is herein defined toencompass both the emission of radiation (yes or no; on or off) as wellas the level of irradiance. In a preferred embodiment, intensity ismonitored at least 4 locations: with at least 2 for each side of theplane of irradiation. In one embodiment, the monitors are photodiodes,each positioned to measure the output of one or more sources ofradiation.

[0083] A preferred source of ultraviolet radiation is a fluorescentsource. Fluorescence is a special case of luminescence. Luminescenceinvolves the absorption of electromagnetic radiation by a substance andthe conversion of the energy into radiation of a different wavelength.With fluorescence, the substance that is excited by the electromagneticradiation returns to its ground state by emitting a quantum ofelectromagnetic radiation. While fluorescent sources have heretoforebeen thought to be of too low intensity to be useful forphotoactivation, in one embodiment the present invention employsfluorescent sources to achieve results thus far achievable on onlyexpensive equipment.

[0084] As used here, “fixed distance” is defined as a constant distancebetween a point in the plane which the means for supporting a pluralityof blood bags defines and a point within the light source. It is knownthat light intensity from a point source is inversely related to thesquare of the distance from the point source. Thus, small changes in thedistance from the source can have a drastic impact on intensity. Sincechanges in intensity can impact photoactivation results, the presentinvention contemplates the use of an extended bar of lamps for a sourceof radiation. Extended bar lamps minimize the effect of small distancechanges on intensity of radiation, providing reproducibility andrepeatability.

[0085] Geometry relates to the positioning of the light source. Forexample, it can be imagined that light sources could be placed aroundthe sample holder in many ways (on the sides, on the bottom, in acircle, etc.). The geometry used in a preferred embodiment of thepresent invention allows for uniform light exposure, of more than onesample, of appropriate intensity for rapid photoactivation. The geometryof a preferred device of the present invention involves multiple sourcesof linear lamps as opposed to single point sources. In addition, thereare several reflective surfaces and several absorptive surfaces.Reflective surfaces can help to even out the exposure of light to eachof a plurality of samples. Because of this complicated geometry, changesin the location or number of the lamps relative to the position of thesamples to be irradiated are to be avoided in that such changes willresult in intensity changes and variability in intensity exposure tomultiple samples.

[0086] Another consideration in obtaining uniform light exposure isprovision for attachments to samples containers during irradiation. Thepresent invention contemplates attachments such as tubing, valves, bloodproduct storage bags, and any other apparatus commonly attached to bagscontaining blood products. This avoids blocking of light by theattachments. In one embodiment, blood bag supporting means has means toposition a plurality of attachments connected to said blood bags, sothat said attachments do not significantly reduce the intensity ofradiation to said blood bags.

[0087] It is useful that an irradiation device deliver the sameintensity of radiation to a sample whether there are several samples orjust a single sample being irradiated at once. The present inventioncontemplates the use of parabolic reflector grids which may bepositioned between the light sources and the sample to be irradiated.These grids direct light passing through, to reduce scatter of light andto avoid decreases in the light impinging on samples when more than onesample is irradiated at the same time.

[0088] In another embodiment, the present invention contemplates the useof a shaking means, such as a shaker or agitator, to mix samples duringirradiation. This mixing may have an averaging effect on the radiationreceived by sample material in different parts of the bag. Withoutintending to be limited by any mechanism by which shaking effectsirradiation predictability, it is contemplated that sample material ismoved throughout the bag during irradiation by shaking, thereby exposingeach part of the sample to many different positions to receiveradiation. If variations in the intensity of radiation exist indifferent areas of the bag, movement would act to reduce the variationin intensity within the sample.

[0089] The present invention further contemplates that the delivery oflight from the light sources will be approximately uniform along thelength of the light source. Some light sources, particularly longtubular bulbs, display a falloff of output at the ends of the bulbs. Theends also tend to give off the most heat. To ensure even illuminationand a controlled temperature, one embodiment of the present inventionhas a lip which wraps around the ends of the light sources to blockapproximately 2-6 cm of the light source on each end from irradiatingsamples in the device.

B. Rapid Photoactivation

[0090] The light source of the preferred embodiment of the presentinvention allows for rapid photoactivation. The intensitycharacteristics of the irradiation device have been selected to beconvenient with the anticipation that many sets of multiple samples mayneed to be processed. With this anticipation, a fifteen minute exposuretime or less is a practical goal. Because sources of ultraviolet lightmay vary in flux over a set amount of time, in a preferred embodiment ofthe present invention, several light output detectors are positionedthroughout the device to measure output of the light sources. In oneembodiment, the detectors are wired to a feedback control, which can beadjusted to shut off the light source when a certain output level hasbeen reached. This ensures repeatability, which is preferable wheninactivating pathogens in blood products. With control of the exposureof light a blood product receives, one can also ensure a sufficientexposure to inactivate pathogens, without having to expose the sample toexcess light, which could be damaging.

[0091] In designing the devices of the present invention, relativeposition of the elements of the preferred device have been optimized toallow for fifteen minutes of irradiation time, so that, when measuredfor the wavelengths between 320 and 350 nanometers, an intensity fluxgreater than approximately 1 mW cm⁻², and preferably 15 mWcm⁻² isprovided to the sample vessels. In a preferred embodiment, the deviceirradiates both sides of the bag.

C. Processing of Large Numbers of Samples

[0092] As noted, another important feature of the photoactivationdevices of the present invention is that they provide for the processingof large numbers of samples. In this regard, one element of the devicesof the present invention is a means for supporting a plurality of bloodproducts, and in particular, blood bags. In the preferred embodiment ofthe present invention the supporting means comprises glass platesbetween two banks of lights with a capacity of six 50 ml bags(equivalent to Dupont Stericell™ bag) plus connectors and tubing, at onetime. By accepting commonly used commercially available blood bags, thedevice of the present invention allows for convenient processing oflarge numbers of samples.

[0093] In a preferred embodiment, the plate has a means to positionattachments connected to said blood bags, such as tubing and satellitestorage bags, so that said attachments do not significantly reduce theintensity of radiation to said blood bags.

D. Temperature Control

[0094] As noted, one of the important features of the photoactivationdevices of the present invention is temperature control. Temperaturecontrol is important because the temperature of the sample at the timeof exposure to light can dramatically impact the results. For example,conditions that promote secondary structure in nucleic acids alsoenhance the affinity constants of many psoralen derivatives for nucleicacids. Hyde and Hearst, Biochemistry, 17, 1251 (1978). These conditionsare a mix of both solvent composition and temperature. With singlestranded 5S ribosomal RNA, irradiation at low temperatures enhances thecovalent addition of HMT to 5S rRNA by two fold at 4° C. compared to 20°C. Thompson et al., J. Mol. Biol. 147:417 (1981). Even furthertemperature induced enhancements of psoralen binding have been reportedwith synthetic polynucleotides. Thompson et al, Biochemistry 21:1363(1982).

[0095] With respect to bacteria, it should be noted that repair ofcrosslinks occurs during irradiation. However, where a lower temperatureis employed during irradiation, the bacterial repair process issuppressed. Thus, a 15° C. irradiation has a significant effect on thelevel of inactivation that is observed.

[0096] Additionally, certain blood preparations can be damaged by smallchanges in temperature. For example, platelets are best preserved ifmaintained at 22° C.±2. Thus it is preferred that a photoactivationdevice for platelets maintain the platelets within or near this rangeduring radiation or the clinical efficacy of the platelets may bereduced.

[0097] Without intending to be limited to any particular means ofcontrolling the temperature of blood products during irradiation on thedevice, in one embodiment the device employs two temperature controllingmeans. A first temperature controlling means is a means for blowing airfrom outside of the housing of the device, across the irradiationproviding means and back out, to cool them and avoid heat transfer toirradiated samples.

[0098] A second temperature controlling means operates in a closedsystem, cooling and circulating air only from within the system, toavoid the recycling of heat carried in air exhausted from the firsttemperature controlling means. The second means is for circulatingcooled air through the air circulation chamber and within the channel inwhich the blood bags rest. This second temperature controlling meansuses a heat exchanger and a means to circulate the cooled air.Preferably, the heat exchanger is a conduit, having an inlet port and anoutlet port for the circulation of temperature control liquid. Theconduit may be covered by a corrugated material with high heatconductivity, which serves to increase the surface area for exchange ofheat between the conduit and the air. In one embodiment, the means tocirculate air is driven by a DC motor, which produces less ambient heatthan an AC motor. Alternatively, the means to circulate air may bedriven from outside the housing. Either alternative controls the amountof heat produced within the housing.

[0099] Cooled air is circulated over the conduit, across the blood bagswhich contain the blood products, and through chambers and channelswhich surround the blood bags. The chambers and channels are also closedoff from significant exchange with air originating from outside thehousing of the device or air passing over the means from providingelectromagnetic radiation, thereby creating “a closed system” thatrecirculates air. In an alternative embodiment, the present inventioncontemplates that the second temperature controlling means comprises arefrigeration unit installed within the housing of the photoactivationdevice.

[0100] Air circulating in the second temperature control means does notmix with air blowing through the first temperature control means. Thisseparation of temperature control allows the samples to be cooledappropriately.

[0101] In one embodiment, the device of the present invention comprisesa shaking means, such as a shaker or an agitator, giving the sampleshorizontal unidirectional and sinusoidal motion of variable frequencyand amplitude. The use of a shaking means during irradiation with thedevice is contemplated for maintaining an even temperature throughoutthe samples within the blood bags by providing mixing of a sample in theblood bags during irradiation. Additionally, use of a shaker forplatelet samples reduces platelet activation during storage. In oneembodiment, a shaker is positioned within the housing of the irradiationdevice, moving the samples by contacting the blood bag supporting meansdirectly. It is contemplated that the top plate of the lower plateassembly may not be fixed with respect to the rest of the lower plateassembly, thus allowing a shaker to contact the top plate directly toacheive agitation. Also contemplated is the movement of the entire lowerplate assembly by a shaker. Alternatively a shaker may be positionedoutside the housing, moving the samples by moving the entire housing ofthe device. The present invention contemplates the use of a ridged uppersurface on the blood bag supporting means which provides frictionsufficient to maintain the position of the sample blood bags duringshaking.

[0102] In another embodiment, heat from the lamps, ballasts and othersources is kept away from the blood bags by one or several partitionbetween the various sources of heat and the bags. This further assistsin maintaining a biologically acceptable temperature in the samples.

E. Inherent Safety

[0103] Ultraviolet radiation can cause severe burns. Depending on thenature of the exposure, it may also be carcinogenic. The light source ofa preferred embodiment of the present invention is shielded from theuser. This is in contrast to the commercial hand-held ultravioletsources as well as the large, high intensity sources. In a preferredembodiment, the irradiation source is contained within a housing made ofmaterial that obstructs the transmission of radiant energy (i.e. anopaque housing). No irradiation is allowed to pass to the user. Thisallows for inherent safety for the user.

F. Sample Containers

[0104] The material of the container which holds the sample to beirradiated in the irradiation device can effect how well the irradiationdevice operates. The material used can effect the penetration ofradiation to the sample and the amount of scatter of radiation impingingon the container. The sample container of one embodiment of the presentinvention is a blood bag made of a plastic transparent to ultravioletlight, preferably Teflon (available from American Fluroseal, SilverSpring, Md.). Some other acceptable plastic components are ethyl vinylacetate (bags available from Terumo, Japan); poly (vinyl chloride) (PVC)(bags available from Baxter Travenol or Cutter, Covina, Calif.), whichmay be combined with plasticisers; or polyolefin (bags available fromthe Fenwal Division of Baxter Travenol Laboratories, Inc., Deerfield,Ill.). For PVC, contemplated placticisers are di (2-ethylhexyl)phthalate (DEHP), tri (2-ethylhexyl) trimellitate (TEHTM). The presentinvention, however, is not intended to be limited to any composition ofblood bag, but contemplates the use of any bag that is somewhattransparant to ultraviolet light. The material may also effectconcentration of components in the sample to be irradiated. In apreferred embodiment, the sample is irradiated on the irradiation devicein a bag that does not bind a significant percent of photoreactivecompound contained in the sample.

[0105] The parameters of the container also control to some extent howthe sample is affected during the irradiation. For example, a blood bagfor platelet storage preserves platelets better if its walls are thinenough to allow the transfer of sufficient oxygen to prevent theincreased rate of lactate production which causes decreased plateletviability. Carmen, R., “The Selection of Plastic Materials for BloodBags,” Transfusion Med. Rev. 7:1 (1993).

[0106] The nature of the blood product may have an impact on efficiency.Red cells, for example, absorb different wavelengths of light than doplatelets. Red cells may reduce the efficiency of irradiation due toblocking of light. Therefore, platelets contaminated with red cells maysee a lower intensity of light than platelet preparations containing nored blood cells.

[0107] As pointed out above, changes in intensity can impactphotoactivation results, and these changes can result from changes indistance within the sample through which the radiation must travel. Thethickness of the sample, as defined by the walls of the blood bag,andthe volume of the blood product, also effect how much light can reachthe sample. In a preferred embodiment of the present invention, when theblood bag containing the sample for irradiation rests within theradiation device, it forms a film of blood product which has a “centralpath length” of between approximately 0.1 and 4 cm. A “central pathlength” is here defined as the shortest distance between two walls of ablood bag that passes through the center of the bag. In one embodiment,the “central path length” of the sample is a fixed value for all bagsused. This provides reproducibility and repeatability. In a preferredembodiment, a shaker is employed, to provide movement, or slushing, ofthe sample material so that each part of the sample is brought to thesurface of the blood bag during irradiation. This may allow forvariation in the central path lengths of the bags, while preservingreproducibility and repeatability, because the agitation may cycle thesample to the surface of the blood bag. Therey, it is ensured thatsufficient light can reach the samples regardless of the central pathlength. In another preferred embodiment, no other pressure need beexerted on the bag by the radiation device, or any other source, otherthan the force of gravity, to obtain the preferred “central pathlength.” In one embodiment, the upper and lower plate assemblies areseparated by between approximately 1 and 10 cm, to accommodate bagshaving a central path length within that range.

EXPERIMENTAL

[0108] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0109] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); HPLC (HighPressure Liquid Chromatography).

EXAMPLE 1

[0110] As noted above, the present invention contemplates devices andmethods for the activation of photoreactive nucleic acid bindingcompounds. In this example, a photoactivation device is described fordecontaminating blood products according to the method of the presentinvention. This device comprises: a) means for providing appropriatewavelengths of electromagnetic radiation to cause activation of at leastone photoreactive compound; b) means for supporting a plurality of bloodproducts at a fixed distance from the radiation providing means duringactivation; and c) means for maintaining the temperature of the bloodproducts within a desired temperature range during activation.

[0111]FIG. 1 is a perspective view of one embodiment of the deviceintegrating the above-named features. The figure shows an opaque housing(100) with a portion of it removed, containing an array of bulbs (101)above and below a plurality of representative blood product containingmeans (102) placed between plate assemblies (103, 104). The plateassemblies (103, 104) are described more fully, subsequently.

[0112] The bulbs (101), which are connectable to a power source (notshown), serve as a source of electromagnetic radiation. While notlimited to the particular bulb type, the embodiment is configured toaccept an industry standard, dual bipin lamp.

[0113] The housing (100) can be opened via a latch (105) so that theblood product can be placed appropriately. As shown in FIG. 1, thehousing (100), when closed, completely contains the irradiation from thebulbs (101). During irradiation, the user can confirm that the device isoperating by looking through a safety viewport (106) which does notallow transmission of ultraviolet light to the user.

[0114] The housing (100) also serves as a mount for several electroniccomponents on a control board (107), including, by way of example, amain power switch, a count down timer, and an hour meter. Forconvenience, the power switch can be wired to the count down timer whichin turn is wired in parallel to an hour meter and to the source of theelectromagnetic radiation. The count down timer permits a user to presetthe irradiation time to a desired level of exposure. The hour metermaintains a record of the total number of radiation hours that areprovided by the source of electromagnetic radiation. This featurepermits the bulbs (101) to be monitored and changed before their outputdiminishes below a minimum level necessary for rapid photoactivation.

[0115]FIG. 2 is a cross-sectional view of the device shown in FIG. 1along the lines of 2-2. FIG. 2 shows the arrangement of the bulbs (101)with the housing (100) opened. A reflecting means (108A, 108B)completely surrounds each array of bulbs (101). Blood product containingmeans (102) are placed between upper (103) and lower (104) ultravioletlight transparent plate assemblies. When the upper plate assembly (103)is lowered over the lower plate assembly (104), the upper (103) andlower (104) plate assemblies define a channel (116—not show in thisfigure) through which air can be circulated to cool the blood productcontaining means. Each plate assembly is comprised of top (103A, 104A)and bottom (103B, 104B) plates. The plate assemblies (103, 104) areconnected via a hinge (109) which is designed to accommodate the spacecreated by the blood product containing means (102). The upper plateassembly (103) is brought to rest just above the top of the bloodproduct containing means (102) supported by the bottom plate (104B) ofthe lower plate assembly (104). In an alternative embodiment, the upperplate assembly (103) may be in a fixed relationship with the housing(100) and the entire top part of the housing, including the upper plateassembly (103) can be brought to rest just above the top of the bloodproduct containing means (102).

[0116] Detectors (110A, 110B, 110C, 110D) may be conveniently placedbetween the plates (103A, 103B, 104A, 104B) of the plate assemblies(103, 104). They can be wired to a printed circuit board (111) which inturn is wired to the control board (107).

[0117]FIG. 3 is a cross-sectional view of the device shown in FIG. 1along the lines of 3-3. Six blood product containing means (102) (e.g.Teflon™ platelet unit bags) are placed in a fix relationship above anarray of bulbs (101). The temperature of the blood product can becontrolled via a fan (112) alone or, more preferably, by employing aheat exchanger (113) having cooling inlet (114) and outlet (115) portsconnected to a cooling source (not shown).

[0118]FIG. 4 is a cross-sectional view of the device shown in FIG. 1along the lines of 4-4. FIG. 4 more clearly shows the temperaturecontrol approach of a preferred embodiment of the device. When the upperplate assembly (103) is lowered over the lower plate assembly (104), theupper (103) and lower (104) plate assemblies define a channel (116)bordered by the bottom plate (103B) of the upper assembly (103) and thetop plate (104A) of the lower assembly (104). Upper plate assemblyplates (103A, 103B) and lower plate assembly plates (104A, 104B) eachdefine an air circulation chamber (103C, 104C), respectively. The fan(112) can circulate air within the chambers (103C, 104C). When the heatexchanger (113) is employed, the circulating air cooled and passedbetween the plates (103A, 103B, 104A, 104B) within the air circulationchambers (103C, 104C), by the fan (112) and then returned to the heatexchanger (113) through the channel (116) between the upper (103) andlower (104) plate assemblies, thereby cooling the blood productcontaining means. The circulating air is kept within a closed systemwhen the housing (100) is in the closed position, comprising the channel(116), the air circulation chambers (103C, 104C) and the surface of theheat exchanger (113) and the fan (112). The air within the closed systemdoes not mix or exchange with the air outside the housing or within thehousing which is not part of the closed system, such as the areasurrounding the bulbs (101).

EXAMPLE 2

[0119]FIG. 5 shows an embodiment wherein platelets are treated by themethod of the present invention. Following fractionation, platelets aretransferred to a bag containing a nucleic acid binding compound (shownin FIG. 1 as a shaded bag). This bag, which has transmission propertiesand other characteristics suited for the present invention, is thenplaced in an irradiation device (such as that described in Example 1,above) and is irradiated. The free compound may be collected or“captured” as desired by a capture device. In such a case, the bag wouldcontain only compound that is contained in cells; the bag would have nofree compound (this bag is indicated in FIG. 1 as unshaded).

EXAMPLE 3

[0120] In this example, the decontamination methods of the presentinvention are applied to inactivate Yersinia enterocolitica, wild type,serotype 3, biotype 4. This organism is found in blood products. Seegenerally R. Y. Dodd, In: Transfusion Medicine in the 1990's (AmericanAssoc. Blood Banks 1990) (S. J. Nance, ed.). See also B. J. Grossman etal., Transfusion 31:500 (1991).

[0121] An overnight culture of the organism was made by inoculating 10ml of brain-heart infusion (BHI) broth from a motility stab. This wasmaintained at 35° C. and 0.1 ml of it was used to inoculate 20 ml of BHIbroth for use in the experiment. After overnight incubation at 35° C.,the stationary culture was pelleted for 15 minutes at 1900 g, thesupernatant was discarded, and the bacterial pellet was resuspended in 1ml of heat-inactivated normal serum pool. This was infused into afreshly expired unit of human platelets obtained from the Blood Bank ofAlameda-Contra Costa Medical Association. 5 ml aliquots of bacteriacontaining platelet concentrate were drawn from the bag and receivedspecified amounts of 8-MOP and UVA irradiation, except for the controls,which were irradiated without psoralen, or received no treatment (seeTable 2). Temperature was maintained at 25° C. during irradiation byplacing the platelet concentrate in stoppered glass water-jacketedchambers attached to a circulating water bath. The irradiation device(Derma Control, Dolton, Ill., Model No. 1224-Special) employed twoarrays (six lamps/array spaced at 2.5 inches), one array above thesample and one bank below the sample (the sample is thus approximately 3inches from the lamps). Each array is separated from the other byapproximately six inches, has a polished metal reflector behind it, andis covered by a UVA-transmitting acrylic plastic sheet. The sample to beprocessed (e.g. platelet bag) sits on the lower sheet. TABLE 2 8-MOP/irr. time drug ml (min) log/ml -titer 1 no drug 0 9.1 2 no drug 10 9.30.2 3 8-MOP 30 ug 10 <0 >−9.1 4 8-MOP 10 10 <0 >−9.1 5 8-MOP 3 10 3.4−5.7 6 8-MOP .2 10 6.8 −2.3 7 8-MOP .06 10 9.0 −0.1

[0122] Derma Control F587T12-BL-HO type bulbs were used. These are“black light” tubes (engineered to emit specific wavelengths by means ofan internal phosphor coating) 24 inches in length. The peak wavelengthis below 360 nm, unlike simple mercury lamps or common “BLB” fluorescentbulbs. Total intensity is less than 20 mW/cm².

[0123] Bacteria were quantified by plating 0.1 ml of serial 10-folddilutions in BHI broth onto 100 mm petri dishes containing BHI agar.After 24 hr incubation at 35° C., colonies were counted and bacterialconcentration was calculated on a per ml basis. The results (Table 2)show that as little as 3 ug/ml of 8-MOP is able to inactivate almost sixlogs of bacteria. With 10 ug/ml, ten minutes provide more than enoughirradiation. Indeed, with 10 ug/ml, five minutes of irradiation appearsto be adequate.

EXAMPLE 4

[0124] Artuc and co-workers examined the solubility of 8-MOP in humanand bovine serum proteins, and showed that at 8-MOP concentrationsranging from 100 to 1000 ng/ml. concentrations similar to those observedin patients undergoing psoralen ultraviolet A (PUVA) therapy forpsoriasis, 75% to 80% of the 8-MOP was bound to albumin. M. Artuc etal., Brit. J. Derm. 101:669 (1979).

[0125] In this example, the binding of 8-MOP to Calf Thymus DNA iscompared using plasma and a protein free medium in order to validate theefficiency of psoralen-nucleic interactions under the decontaminationmethods of the present invention. Although this measurement usedeukaryotic nucleic acid rather than bacterial nucleic acid, it is auseful indicator of the degree of adduct formation for bacteria.

[0126]³H-8-MOP was prepared to a concentration of 115 ug/ml in ethanolat a specific activity of 4.7×10⁶ CPM/microgram (hereinafter “8-MOPstock”). Thereafter 130.5 or 22 ul of 8-MOP stock (2 each) for samplescontaining DNA (“+DNA”) and 52.2 or 8.7 ul for samples not containingDNA (“−DNA”) were dried down. To +DNA samples, 40 ul of DNA stock (7.7mg/ml) was added as well as either 460 ul plasma (day old frozen) or 450ul Tris-EDTA (“TE”) buffer. To the latter was also added 10 ul 5M NaCl.For—DNA samples (i.e. the controls), 184 ul plasma and 16 ul water wasadded.

[0127] The samples were mildly vortexed for approximately one hour andthe counts were checked to confirm that the 8-MOP dissolved.

[0128] Each sample (100 ul) was irradiated on an HRI-100 (HRI ResearchInc., Concord, Calif.) at 25° C. for 0, 2, 4, 8, and 16 minutes. Sampleswere kept at 4° C. overnight after irradiation. Thereafter, the sampleswere extracted. First, a phenol solution was prepared at pH 8 byequilibrating with 0.1 M Tris pH 8. Each sample was then extracted with100 ul phenol. Each sample was centrifuged for 5 minutes to remove theaqueous phase to a new tube. A second extraction was performed with 100ul 1:1 phenol:chloroform. A final extraction was performed with 100 ulchloroform.

[0129] The final aqueous phase was precipitated by adding 50 ul NaCladjusted to give a final concentration of NaCl of 0.2 M and then adding250 ul ethanol. The samples were again centrifuged (10 minutes). Thesupernatant was removed and the pellets were dried. The pellets wereresuspended in 100 ul TE and re-precipitated. This was repeated for atotal of 3 precipitations. The final pellets were brought up in 600 ulwater and 100 ul was counted. Each sample was assayed for DNA bymeasuring absorbency (260 nm). 8-MOP levels were plotted as adducts per1000 base pairs (“8-MOP:kBP”).

[0130] The results (FIG. 6) show that plasma does significantly changethe addition kinetics of 8-MOP to DNA. Addition to nucleic acid is muchbetter in the protein free media.

[0131] The frequency of 8-MOP-DNA adduct formation in protein free mediapredicts a high multiplicity of modification of the bacterial genome.Furthermore, this type of biochemical measurement has the potential toprovide a means to monitor the efficiency of the photochemicalinactivation method.

EXAMPLE 5

[0132] Photoactivation of psoralens and isopsoralens may result in avariety of photoproducts. “Photoproduct” is best understood byconsidering the possible reactions of photoreactive compound whenexposed to activating wavelengths of electromagnetic radiation. Whilenot limited to any precise mechanism, it is believed that the reactionof photoreactive compound in its ground state (“C”) with activatingwavelengths of electromagnetic radiation creates a short-lived excitedspecies (“C*”):

C→C*

[0133] What happens next is largely a function of what potentialreactants are available to the excited species. Since it is short-lived,a reaction of this species with nucleic acid (“NA”) is believed to onlybe possible if nucleic acid is present at the time the excited speciesis generated. Thus, the reaction must, in operational terms, be in thepresence of activating wavelengths of electromagnetic radiation, i.e. itis “photobinding”; it is not dark binding. The reaction can be depictedas follows:

C*+NA→NA:C

[0134] The product of this reaction is hereinafter referred to as“Photoaddition Product” and is to be distinguished from “Photoproduct.”

[0135] With this reaction described, one can now consider the situationwhere nucleic acid is not available for binding at the time the compoundis exposed to activating wavelengths of electromagnetic radiation. Sincethe excited species is short-lived and has no nucleic acid to reactwith, the excited species may simply return to its ground state:

C*→C

[0136] On the other hand, the excited species may react with itself(i.e. a ground state or excited species) to create a ground statecomplex (“C:C”). The product of these self-reactions where two compoundsreact is referred to as “photodimer” or simply “dimer.” Theself-reactions, however, are not limited to two compounds; a variety ofmultimers may be formed (trimers, etc.).

[0137] The excited species is not limited to reacting with itself. Itmay react with its environment, such as elements of the solvent (“E”)(e.g. ions, gases, etc.) to produce other products:

C*+E→E:C

[0138] It is this type of reaction that is believed to cause cellulardamage (e.g., reaction with oxygen to create singlet oxygen species).Furthermore, it may simply internally rearrange (“isomerize”) to aground state derivative (“[”):

C*→[

[0139] Finally, the excited species may undergo other reactions thandescribed here.

[0140] The present invention and the understanding of “photoproduct”does not depend on which one (if any) of these reactions actuallyoccurs. “Photoproduct”—whatever its nature—is deemed to exist if,following the reaction of a compound and activating wavelengths ofelectromagnetic radiation, there is a resultant product formed that caninteract with other components of the reaction environment.

[0141] With psoralens such as 4′-hydroxymethyl-4,5′,8-trimethylpsoralen(HMT), there is a number of resultant products produced when the HMT isexposed to activating wavelengths of electromagnetic radiation. Themajor resultant products of HMT are two cyclobutyl photodimers. In oneof the dimers, the two pyrone rings are linked in a cis-synconfiguration, while in the other dimer, the linkage occurs between thefuran end of one molecule and the pyrone end of the other, again withcis-syn configuration. A third resultant product of HMT is a monomericHMT photoisomer. In this isomer, the central ring oxygens assume a 1, 4instead of the normal 1, 3 orientation. While the two photodimers wouldnot be expected to have an intercalating activity due to geometricalconsiderations, the photoisomer remains planar, and accordingly, it iscontemplated that it has a positive intercalative association withdouble stranded nucleic acid and, thus, could be a mutagen.

[0142] In this example, the photochemical breakdown of 8-MOP is comparedwith AMT. The samples were analyzed by reverse phase HPLC using a RainenDynamax 300A column. Gradient elution was performed with 0.1 M ammoniumacetate/acetonitrile (0-70% acetonitrile over 42 minutes). AMT elutes asa single peak at approximately 24 minutes under these conditions.Detection was by absorption at either 260 or 330 nm. The latterwavelength was used for the plasma containing samples.

[0143] Standard solutions of each compound were prepared at variousconcentrations. These solutions were then diluted 1:10 into water, then300 ul injected for analysis. All samples were monitored at 300 nm.Peaks were analyzed by measuring either peak height or peak area, thenconverted to a gh/ml value using the standard plot. Peak area wasdetermining by photocopying the trace, cutting out the copy of the peak,then weighing the resultant trace. The two methods gave essentially thesame result.

[0144] The results are shown in FIG. 7. Clearly, AMT degrades morequickly than 8-MOP. It would, therefore, be expected to generate morephotoproducts—which eventually would end up in the transfusionrecipient. By contrast, it is not expected that 8-MOP generates asignificant amount of photoproducts. This is important when oneconsiders that the weight of authority has concluded that non activated8-MOP is not mutagenic.

EXAMPLE 6

[0145] When platelets become activated, an alpha granule membraneglycoprotein called GMP140 becomes exposed on the platelet surface. Lessthan (5%) of fresh, normal unstimulated platelets express detectable GMP140 levels by flow cytometry. See generally M. J. Metzelaar, Studies onthe Expression of Activation-Markers on Human Platelets (Thesis 1991).

[0146] To measure GMP140, a small aliquot of platelet rich plasma isplaced in HEPES buffer containing a GMP140-binding antibody or controlmouse IgG. CD62 is a commercially available monoclonal antibody whichbinds to GMP 140 (available from Sanbio, Uden, the Netherlands; CaltagLabs, So. San Francisco, Calif., and Becton Dickinson, Mountain View,Calif.). After a fifteen minute incubation, Goat Anti-Mouse IgGconjugated to FITC is added to the tube in saturating amounts. Finally,the cells are diluted in isotonic saline, fixed with paraformaldehydeand analyzed on a FACSCAN™ (Becton Dickinson, Mountain View, Calif.).The positive control is made by adding Phorbol Myristate Acetate (PMA)to the test system at a final concentration of 10−⁷ M.

[0147] In this example, CD62 was employed to measure the impact, if any,of irradiation alone on platelet activation. The antibody was stored insmall aliquots (0.01 mg/ml) at −40° C. prior to use. A mouse IgG control(0.05 mg/ml) (Becton Dickinson, Mountain View, Calif. #9040) 5×concentrated was employed. At time of use, this was diluted 1:5 in HEPESbuffer. The secondary antibody was goat Anti-Mouse IgG conjugated toFITC (TAGO, Burlingame, Calif. #3506). This was stored in small aliquotsat −20° C. Phorbol Myristate Acetate (PMA) (Sigma, St. Louis, Mo.) wasstored at −40° C. At time of use, this was dissolved in DMSO (workingconcentration was 1.62×10−⁵ M).

[0148] 16% Paraformaldehyde (PFA) (Sigma, St. Louis, Mo.) was preparedby adding 16 grams paraformaldehyde to 100 ml de-ionized water. This washeated to 70° C., whereupon 3 M NaOH was added dropwise until thesolution was clear. The solution was cooled and the pH was adjusted to7.4 with 1 N HCl. This was filtered and stored. A commercially availableisotonic buffer was used: Hematall Isotonic Diluent (Fisher #CS 606-20).

[0149] For measuring platelet activation of platelet concentrates, aunit of human platelets was obtained from the Blood Bank ofAlameda-Contra Costa Medical Association. 5 ml aliquots were drawn fromthe bag and received specified amounts of UVA irradiation, except forthe control, which received no treatment other than being placed in achamber for irradiation. Temperature was maintained at 25° C. duringirradiation by placing platelet concentrate in stoppered glasswater-jacketed chambers attached to a circulating water bath. Theirradiation device (Derma Control, Dolton, Ill.; Model No. 1224-Special)was as described in Example 3, above. Following irradiation, theplatelets were stored for 5 days. At specific time points, aliquots weretaken and processed.

[0150] Processing involved adding an aliquot (e.g. 5 microliters) ofplatelet concentrate to each microcentrifuge tube containing theantibody and appropriate reagents and this was mixed very gently byvortex. The samples were incubated for 15 minutes at room temperature.

[0151] The Goat anti-Mouse IgG-FITC (diluted 1:10 in HEPES buffer) wasadded (5 microliters) to each tube and the solution was mixed by gentlevortex. The samples were incubated for an additional 15 minutes at roomtemperature.

[0152] Isoton II was added (1 ml) to each tube and mixed gently with apolypropylene disposable pipette. 8% PFA in HEPES (150 microliters) wasadded to each diluted sample to final 1%. The platelets were analyzed onthe FACSCAN™. The results are shown in Table 3. TABLE 3 Day 3 Day 5 PMAPMA Conditions Unactivated Activated Unactivated Activated Control 17 8525 89 UV 5′ 17 87 24 86 UV 10′ 51 84 77 79

[0153] Activation is expressed as a percent. Clearly, irradiation forten minutes (UV 10′) resulted in a significant negative impact on storedplatelets; the platelets were highly activated. By contrast, irradiationfor five minutes (UV 5′) resulted in no significant activation above thecontrol which received no irradiation.

EXAMPLE 7

[0154] Given the results of Example 6, it is clear that either a shorterirradiation time or the use of filters is needed to avoid damage tocells by UV irradiation. In this example, CD62 is employed to measurethe impact of irradiation in the presence of psoralen on plateletactivation. Shorter irradiation times and wavelength filters areseparately employed.

[0155] Shorter Irradiation Times. A unit of human platelets was againobtained from the Blood Bank of Alameda-Contra Costa MedicalAssociation. 5 ml aliquots were drawn from the bag to receive fiveminutes (5′) of UVA irradiation in the presence of 10 ug/ml 8-MOP,except for the control, which received no treatment other than beingplaced in a chamber for irradiation. Temperature was maintained at 25°C. during irradiation by placing platelet concentrate in stoppered glasswater-jacketed chambers attached to a circulating water bath. Theirradiation device (Derma Control, Dolton, Ill.; Model No. 1224-Special)was as described in Example 3, above.

[0156] Following irradiation, the platelets were again stored for 5 daysas in Example 6. At specific time points, aliquots were taken andassayed with the CD62 antibody and analyzed on the FACSCAN™ to showthat, under these conditions, platelets can be inactivated withoutdamage to the cells and stored for five days prior to transfusion.

[0157] Wavelength Filters. An aqueous solution of Co(No₃)₂ was used incombination with NiSO₄ to substantially remove the 365 nm component ofthe emission spectrum of the light source employed. The Co—Ni solutioncan be conveniently used in place of water as a coolant during theirradiation.

[0158] Following a ten minute irradiation with the filter, the plateletswere stored an assayed with the CD62 antibody on the FACSCAN™ to showthat, under these conditions, platelets can be inactivated withoutdamage to the cells and stored for five days prior to transfusion.

[0159] The above has been offered for illustrative purposes only, and isnot intended to limit the scope of the invention of this application,which is as defined in the claims below.

We claim:
 1. A blood treatment device, comprising: i) a fluorescent bulb; ii) a light filtration material configured to remove a blood damaging wavelength of electromagnetic radiation emitted by the fluorescent bulb to produce filtered electromagnetic radiation; and iii) a blood bag support configured such that at least a portion of the filtered electromagnetic radiation from the bulb contacts a blood bag when positioned on the support.
 2. The device of claim 1, wherein the material removes a blood product damaging wavelength that is absorbed by a nucleic acid.
 3. The device of claim 1, wherein the material removes a blood product damaging wavelength that is absorbed by a protein.
 4. The device of claim 1, wherein the material removes a blood product damaging wavelength that is absorbed by hemes.
 5. The device of claim 1, wherein the material removes blood product damaging wavelengths that are absorbed by a nucleic acid, a protein and hemes.
 6. The device of claim 5, wherein said filtered electromagnetic radiation has wavelengths between 320 and 400 nm.
 7. The device of claim 1, wherein the blood bag support comprises said light filtration material, and said light filtration material comprises a short wavelength cutoff filter.
 8. The device of claim 7, wherein the short wavelength cutoff filter is a 320 nm cutoff filter.
 9. The device of claim 8, wherein said filtered electromagnetic radiation has wavelengths between 320 and 400 nm.
 10. The device of claim 1, wherein said filtered electromagnetic radiation has a wavelength that is absorbed by flavins.
 11. A method of treating a blood product in a blood bag, comprising: a) providing i) a blood bag comprising a photoreactive compound and a blood product suspected of containing a pathogen and ii) a blood treatment device comprising a fluorescent bulb, a light filtration material configured to remove a blood damaging wavelength of electromagnetic radiation emitted by the fluorescent bulb to produce filtered electromagnetic radiation, and a blood bag support configured to receive the filtered electromagnetic radiation; b) positioning the blood bag on the blood bag support; and c) irradiating the blood bag with the filtered electromagnetic radiation so as to activate the photoreactive compound and inactivate the pathogen.
 12. The method of claim 11, wherein the light filtration material removes a blood product damaging wavelength that is absorbed by a nucleic acid and a protein.
 13. The method of claim 12, wherein the material removes blood product damaging wavelengths that are absorbed by a nucleic acid, a protein and hemes.
 14. The method of claim 13, wherein said filtered electromagnetic radiation has wavelengths between 320 and 400 nm.
 15. The method of claim 11, wherein the photoreactive compound is a psoralen.
 16. The method of claim 15, wherein the blood product comprises plasma.
 17. The method of claim 16, wherein the blood product comprises platelets.
 18. A method of treating a blood product in a blood bag, comprising: a) providing i) a blood bag comprising a photoreactive compound and a blood product suspected of containing a pathogen and ii) a blood treatment device comprising a fluorescent bulb and a blood bag support, wherein the end of the fluorescent bulb is blocked from irradiating the blood product; b) positioning the blood bag on the blood bag support; and c) irradiating the blood bag with the fluorescent bulb so as to activate the photoreactive compound and inactivate the pathogen.
 19. The method of claim 18, wherein the photoreactive compound is a psoralen.
 20. The method of claim 19, wherein the blood product comprises plasma.
 21. The method of claim 20, wherein the blood product comprises platelets. 